The invention relates to the field of combining free electrophoresis and multistage extraction in an instrument capable of separating living cells, particles, proteins, and solutes in useful quantities at high concentrations.
Conventional particle separation techniques typically include centrifugation, which is limited due to its specificity, capacity, speed, energy consumption, biological impact, and microgravity environment disturbances.
Electrophoresis is a leading method for resolving mixtures of cells or charged macromolecules (proteins and nucleic acids). The electrophoretic separation of proteins without gels has been a long-standing goal of separations research. The process of electrophoresis has so far been unable to xe2x80x9cgraduatexe2x80x9d from an analytical tool to a viable unit operation. This is primarily because of various problems such as thermal convection, electro osmosis, particle sedimentation, droplet sedimentation, particle aggregation, and electro hydrodynamic zone distortion have been found to be major obstacles to scale-up. The traditional approach has been to devise density gradients or elaborate flowing devices to counteract these problems; however incorporation of such methods has been unable to address these problems effectively. Also, their addition has caused the process to become cumbersome, thus further reducing the appeal of electrophoresis.
Without the need to prepare density gradients and/or use elaborate flowing systems, free electrophoresis can enjoy much more widespread use because it is a high-resolution method that does not require adsorption to solid media and the subsequent solids handling. It can separate both particles (cells) and solutes (macromolecules) with equal ease. Some specific applications for electrophoresis include the separation of different cells of peripheral blood and bone marrow in hematological and immunological research. Other potential applications include clinical therapeutics and the separation of proteins from body fluids, tissue extracts and fermentation broths.
A mixing problem encountered during free electrophoresis is the mixing caused by the release of gases at the electrodes. However, the use of either non-gassing electrodes such as described in (Agarwala 1994) and incorporated herein or membrane-separated electrodes such as described in (Cole et. al. 1995) and incorporated herein will effectively solve this problem. Experiments performed using palladium electrodes have demonstrated our ability to solve this problem.
Free electrophoresis is a process in which a sample is introduced into a liquid buffer, static or flowing, in a zone and subjected to an electric field in which separands migrate according to their surface charge properties. Fluid instabilities result because longer migration paths are exposed for longer times to developing instabilities.
Timmerman A. Tulp and M. G. Barnhoorn in Preparative Density Gradient electrophoresis of Cells and Cell Organelles A New Separation Chamber, Electroporesis (1982), teaches that a short electrophoretic migration path in a non-moving buffer avoids exposure of migrating separands to unstable buffer flows. Tulp designed a reorienting, free electrophoresis device consisting of a flat disk-shaped container with thin sample bands and a short vertical migration distance. The bottom and the top electrode fluids served as the coolant, the total height of the separation column was 1-2 cm, and its diameter was greater than 15 cm. The distance between the unrelated separands was 1-2 mm, and this distance was increased during fractionation after electrophoresis by re-orienting the disk so that it became a narrow vertical column.
In a different field of separations, Albertsson et al. teaches that multistage extraction processes can proceed in a multistage separator consisting of two sets of cavities facing each other around the periphery of a pair of plates. Further, conventional electrophoresis devices rely on the use of gels, paper or flowing channels to stabilize the electrophoresis buffer in which separands migrate. The devices are limited in capacity and, in the case of flowing channels, difficult for the user to operate and maintain. Obviously, prior art is not meeting the needs of separations by free electrophoresis. Further, applications of free electrophoresis in low gravity require a gravity independent means of collecting electrophoretically separated fractions of the sample.
The present invention fulfills this requirement and is hence ideal for applications in space-flight electrophoresis experiments and applications. For instance, the overall efficacy of electrophoresis as a unit operation can be greatly improved if the migration distance is greatly reduced and the process is multistaged.
A thin-layer countercurrent distribution apparatus is designed and constructed so that up to 20 fractions can be collected on the basis of electrophoretic mobility by application of an electric field. The multistage electrophoretic separation and purification of cells, particles, proteins, and solutes utilize an innovative purification method that combines free electrophoresis and multistage extraction in an instrument capable of separating and/or purifying living cells, particles and proteins in useful quantities and at high concentrations. The mixture to be separated starts in a bottom cavity, and successive top cavities, collect fractions as separand particles or molecules are electrophoresis upward out of the bottom cavity. Mathematical models of this process have been developed, and experiments performed to verify the predictions of the models by collecting and counting particles in each cavity after fractionation. The process depends on the electrophoretic mobility of separands, and is gravitationally stabilized so that it functions in laboratories on earth and in space.
Moreover, an electrophoresis device is disclosed which separates cells, particles, proteins and other separands by collecting samples of decreasing electrophoretic mobility in a train of inverted cavities while an electric field is applied between said inverted cavities and one or more sample cuvettes containing a mixture of cells, particles, proteins or other separands. One circular plate is provided for the one or more sample cuvettes, and one circular plate is provided for the multiple collection cavities. The invention utilizes an innovative purification method that combines free electrophoresis and multistage extraction in an instrument capable of separating living cells, particles, and proteins in useful quantities at high concentrations. The purification method includes a method for dealing with electrolysis products, a technique for controlling the electrical energy input, and an approach for keeping the process isothermal. The invention solves many separation applications problems on earth and also in reduced gravity in space flight.
The multistage electrophoretic purification of cells, particles, and proteins, utilizes an innovative purification method that combines free electrophoresis and multistage extraction in an instrument capable of separating living cells, particles and proteins in useful quantities and at high concentrations. The isothermal process depends on the electrophoretic mobility of separands, and is gravitationally stabilized so that it functions in laboratories on earth and in space. The purification method includes a method for dealing with electrolysis, a technique for transporting and varying electrical energy, and an approach for keeping the process isothermal. The electrophoretic technology resolves many unique separation applications on earth as well as in reduced gravity environments in space flight.
The instant invention is extremely well suited to immunological research, pharmaceutical delivery, biomedical applications, cell biology, and cell separation problems associated with clinical, animal, and plant research. The separation process is well suited to space flight, specifically for on-orbit cell separation problems associated with biological research. Moreover, the electrophoretic technology and electrokinetic separation employ affinity partitioning and electrophoresis. The invention incorporates both features, a short migration distance, and a multistage operation technique in order to increase the throughput of the process and to make the process easier to operate.
The multistage electrophoresis separation and purification assembly utilizes oppositely charged electrodes at the ends of two cavities providing the electric driving force for the migration of particles. It provides a thin layer countercurrent distribution apparatus capable of collecting up to 22 fractions by applying an electric field. The hardware is a combination of free electrophoretic and multistage extraction and consists of 20 or more cavities of a multistage thin layer extraction system. Half cavities oppose each other in disks that are sealed together and one disk rotates with respect to each other. The mixture to be separated starts in a single cavity on a first plate, and successive cavities collect fractions as separand particles or molecules are electrophoresed upward out of the cavity of a second plate. The half cavities are disk shaped, the top cavities having flat tops and the bottom cavities having flat bottoms. Both consist of palladium metal electrodes that produce an electric field when the two cavities re in contact. Each half cavity is only a fee millimeters in height so that the fluid within it remains isothermal during the application of an electric field that transfers separand particles or molecules from the cavity of one plate to the cavity of the corresponding plate.
As each separand is transferred to a new cavity it is swept into the upper half by the electric field or left in the lower half, depending on its electrophoretic mobility. The first fractions collected into the top cavity consist of high mobility separands while later fractions consist of lower mobility solutes or particles. The resulting fractogram corresponds linearly to an electrophoretic mobility distribution. The unit can be operated in various modes such as skimming separands from the top of a single bottom cavity without mixing, or following a true counter-current separation with or without remixing at each stage. A mathematical model, from which distributions and resolution can be derived was formulated, and its predictions tested in multistage experiments.
It is an object of the invention to provide a device for the successful electrophoretic separation of cells, particles, proteins and other separands
It is an object of the present invention to provide a temperature control system capable of controlling the temperature from xe2x88x9237xc2x0 C. to 20xc2x0 C. and preferably at about 4xc2x0 C.
It is an object of the present invention to provide a sample collection capability of one or more independent samples.
It is an object of the present invention to provide a means for holding the magnitude of the electric field is held constant at a selected field strength by a microprocessor-controlled electric circuit,
It is an object of the present invention to provide a means of collecting different types of cells or to collect only cells or particles, only media, or both.
It is an object of the present invention to provide a capability to separate cells from culture medium for sampling.
It is an object of the present invention to provide a medium which is replenishable by means of perfusion which is programmable or active on demand.
It is an object of the present invention to provide as an option a electromagnetic stirring system.
It is an object of the present invention to provide a means of providing the apparatus in a modular cassette in order to facilitate sequential experiments.
It is an object of the present invention to provide a means for the researcher to have experiment flexibility and select solutions, temperatures, and sampling times.
It is an object of the present invention to provide a means for collecting samples of cells, particles, or medium in bags, cuvettes, syringes, or other vessels.
It is an object of the present invention to provide a purification process in which a low conductivity separating buffer is used and electrode metals are selected to prevent gas bubble release and minimize or eliminate the need for active cooling.
It is an object of the present invention to provide a high conductivity separating buffer and receiving electrode metals are selected to prevent gas bubble release, and the one feed cavity electrode is separated from the feed sample cavity by an ultrafiltration membrane and contains an electrolyte that is pumped therethrough.
It is an object of the present invention to provide a multistage electrophoretic separation and purification system designed to be utilized in a cassette integrated within an space flight processing facility.
These and other objects of the present invention will be more fully understood from the following description of the invention.
A preferred embodiment of the present invention provides a multistage electrophoretic purification process for separating and purifying cells, particles, and proteins, and includes a frame and opposing circular plates defining a stationary sample plate containing particles to be separated in a fluid and a rotating collection plate in cooperative sealable engagement with one another, each one including at least one cavity alignable with one another. At least one stationary sample plate cavity and at least one rotating collection plate cavity are positionable for fluid communication with one another arranged to form a multi-stage thin-layer extraction system. At least one stationary sample plate cavity and at least one collection plate cavity contains a metal electrode for producing an electric field in the fluid subjecting the particles to be separated to the electric field separating the particles by their degree of electrophoretic mobility causing migration of the particles and collecting the particles in at least one collection cavity. Upon complete separation the electric field is de-energized and the plates are rotated countercurrently until the upper cavity aligns with a lower cavity with fresh solution that is thoroughly mixed with the separated cells or molecules, and the process is repeated as many times as necessary to effect the desired separation.
A preferred method of separating cells, particles, proteins and other separands with an electrophoresis device comprises the steps of placing a sample of particles to be separated comprising cells, media, proteins or other separands, or mixtures thereof into at least one sample container containing an fluid and supported by means for holding. Rotate a collection plate having at least one collection cavity in sealed cooperative engagement with the at least one sample container. Align the at least one collection cavity with the at least one sample container providing fluid communication therewith. Apply an electric current field to the sample in the at least one sample container while the at least one collection cavity is in fluid communication therewith. Collect a fraction of the sample containing particles to be separated having a different electrophoretic mobility in the at least one collection cavity while the electric field is applied thereto.